Localization of ribosomal DNA within the proximal X heterochromatin of Sciara coprophila (Diptera,Sciaridae)

By means of several reciprocal translocations in Sciara coprophila, each having a break-point in the proximal X heterochromatin, it has been possible in the salivary gland nucleus to bring about separation of specific regions of this heterochromatin and then, by means of in situ hybridization, to determine the relative number of ribosomal RNA cistrons in each. The three blocks of heterochromatin delineated by the translocation break-points have been designated H1, H2, and H3; H1 is the most proximal, lying immediately to the right of the X centromere, and H3 is the most distal, constituting the very end (right) of the chromosome. The distribution of ribosomal RNA cistrons is as follows: 10% are located in H1; 50% in H2; and 40% in H3. For the first time it has been possible to confirm by grain count our previous biochemical estimate of a 60% deletion of rRNA cistrons in the proximal heterochromatin of the X _W homologue. The grain count data also support the conclusion of our previously published cytological analysis, that the exchange points in the X heterochromatin are identical in translocations T29 and T32 (between H1 and H2), also in translocations T23 and T70 (between H2 and H3). The coincidence of break-points in the X heterochromatin is considered in relation to the chromomere make-up of the region. Also, the occurrence of ribosomal RNA cistrons in all three heterochromomeres is discussed in relation to the functional significance of chromomeres.     

X heterochromatin subdivision and cytogenetic analysis in Sciara coprophila (diptera,sciaridae)

The so-called controlling element (CE), which normally programs the curious behavior of the sex chromosome in this genus, has been localized in the short right arm of the polytene X in S. coprophila. The localization was accomplished by use of five X-autosome translocations whose break points define three blocks of heterochromatin (heterochromomeres) extending from the X centromere to the very end (right) of the chromosome. The behavior of the translocation chromosomes at the crucial second spermatocyte division was examined and the precocious chromosome identified in all five cases. Then, knowing the heterochromomere make-up of each chromosome, the position of the CE could be mapped; it is located in heterochromomere H2, the same block of heterochromatin that contains 50% of the ribosomal RNA cistrons. — The question of whether the CE can manipulate any centromere in the nucleus has been only partially answered. It can manipulate translocation chromosomes which possess the centromere of the metacentric autosome (salivary chromosome IV) or that of the shorter rod (salivary chromosome II); but the longer rod (salivary chromosome III) whose proximal end, as seen in the polytene nucleus, is heavily laden with heterochromatin of its own, has not been brought under CE control. — In one of the translocations, T23, the precocious chromosome is a very large metacentric chromosome which resembles the peculiar V-shaped X of S. pauciseta. This peculiarity is not observed in the J-shaped precocious chromosome of T29. These points are discussed.Dedicated to Professor Hans Bauer on the occasion of his 75th birthday.     

The effect of X chromosome heterozygosity on the structure of the ribosomal genes in Drosophila melanogaster.

Sucrose gradient analysis of DNA isolated from detergent-pronase lysates of adult flies has been used to look for ribosomal genes not integrated into the DNA of the chromosome in genotypes containing various combinations of inversions having breakpoints in the proximal heterochromatin of the X chromosome. Unintegrated genes are found in females heterozygous for inversions which have one breakpoint between the nucleolus organizer and the centromere. Homozygotes and males do not have unintegrated genes. The results suggest that unintegrated ribosomal genes result from an interaction between homologues having different arrangements of the proximal heterochromatin. In addition, data from a series of stocks carrying duplications of the X heterochromatin provide independent evidence for the size of the DNA on our gradients.     

Most Uv-Induced Reciprocal Translocations in SORDARIA MACROSPORA Occur in or near Centromere Regions

In fungi, translocations can be identified and classified by the patterns of ascospore abortion in asci from crosses of rearrangement x normal sequence. Previous studies of UV-induced rearrangements in Sordaria macrospora revealed that a major class (called type III) appeared to be reciprocal translocations that were anomalous in producing an unexpected class of asci with four aborted ascospores in bbbbaaaa linear sequence (b = black; a = abortive). The present study shows that the anomalous type III rearrangements are, in fact, reciprocal translocations having both breakpoints within or adjacent to centromeres and that bbbbaaaa asci result from 3:1 disjunction from the translocation quadrivalent.-Electron microscopic observations of synaptonemal complexes enable centromeres to be visualized. Lengths of synaptonemal complexes lateral elements in translocation quadrivalents accurately reflect chromosome arm lengths, enabling breakpoints to be located reliably in centromere regions. All genetic data are consistent with the behavior expected of translocations with breakpoints at centromeres.-Two-thirds of the UV-induced reciprocal translocations are of this type. Certain centromere regions are involved preferentially. Among 73 type-III translocations, there were but 13 of the 21 possible chromosome combinations and 20 of the 42 possible combinations of chromosome arms.     

A ‘zebra’ chromosome arising from multiple translocations involving non-homologous chromosomes

An alloplasmic wheat line carrying a zebra chromosome z5A was isolated from the derivatives of an Elymus trachycaulus x Triticum aestivum cv Chinese Spring hybrid. Chromosome z5A was named zebra because of its striped genomic in situ hybridization pattern. z5A consists of four chromosome segments derived from E. trachycaulus and four chromosome segments, including the centromere, from wheat. The short arm of z5A paired with the telocentric chromosome 1H^tS of E. trachycaulus and the long arm with the long arm of normal 5A. z5A also carried several genetic markers derived from 1H^tS. Chromosome 1H^t was the only E. trachycaulus chromosome found in the sib plants of a previous generation from which z5A was derived. Monosomic 5A and telocentric chromosome 5AL were also found in most of the sib plants. The zebra chromosome most probably originated from spontaneous multiple translocations between chromosomes 5A and 1H^tS or 5A and 1H^t.     

Redundant segments inZea mays detected by translocations of monoploid origin

Twenty-two independently occurring spontaneous reciprocal translocations were isolated from monoploid X diploid crosses in maize and their breakpoints were determined. As 12 of the translocations involved the same two chromosomes and had breakpoints at approximately the same positions (6L. 2–3, 7L. 2–3) and two other translocations appeared to be identical with breakpoints at 2L. 9, 6L. 4, 14 of the 22 translocations probably arose by crossing over within duplicate segments of nonhomologous chromosomes. Thus, at least part of the bivalents seen at diakinesis and chromatid bridges seen at anaphase I in monoploid plants appear to be generated by recombination between redundant chromosome segments. The other eight translocations each occurred once. Because our evidence indicates that recombination between nonhomologous illegitimately synapsed chromosome segments does not occur in maize, these were probably also produced by recombination between redundant segments. If one assumes that their breakpoints also mark regions of interchromosomal redundancy, other conclusions can be reached: A) corn does not contain detectable homoeologous chromosomes, thus it is precently a true diploid, and B) as exchanges giving rise to translocations did not occur in the centromeres or proximal heterochromatin, these regions either do not possess redundancy or are rarely involved in chiasma formation. Furthermore, the duplicated segments in the genome giving rise to translocations in haploid microsporocytes probably have the same serial order with respect to the centromere.This work was partially supported by U.S. Atomic Energy Commission Contract AT(11-1)-2121.     

The location of ribosomal cistrons (rDNA) in chromosomes of the rat

In situ hybridization of ³H-labelled ribosomal RNA to the chromosomes of rat bone marrow cells revealed that clusters of ribosomal cistrons (rDNA) are located in the secondary constrictions of chromosomes No. 3 and 12 and near the centromere of chromosome No. 11, both associated with the late DNA-replicating regions. They were not found in Nos. 1, 2, 13, 19, 20, and the Y chromosome.     

Genetically Induced Mitotic Exchange in the Heterochromatin of DROSOPHILA MELANOGASTER

Multiple copies of the 18S and 28S ribosomal RNA cistrons are present in both the X and Y chromosomes of Drosophila melanogaster. Data are presented here that identify a locus, Rex, that causes exchange-like events between duplicated ribosomal complexes at the ends of an attached-XY chromosome. Rex: (1) is close to or in the basal heterochromatin of the X chromosome; (2) is semidominant and (its effect) is temperature sensitive; (3) acts maternally; and (4) affects behavior of paternally derived attached-XY chromosomes shortly after fertilization. Though, at this point, the existence of Rex is known only from its effects on behavior of a particular compound chromosome, it presents intriguing possibilities for understanding regulation of chromosome behavior and organization of the ribosomal cistrons.     

The Genetic and Cytological Organization of the Y Chromosome of DROSOPHILA MELANOGASTER

Cytological and genetic analyses of 121 translocations between the Y chromosome and the centric heterochromatin of the X chromosome have been used to define and localize six regions on the Y chromosome of Drosophila melanogaster necessary for male fertility. These regions are associated with nonfluorescent blocks of the Y chromosome, as revealed using Hoechst 33258 or quinacrine staining. Each region appears to contain but one functional unit, as defined by failure of complementation among translocations with breakpoints within the same block. The distribution of translocation breakpoints examined appears to be nonrandom, in that breaks occur preferentially in the nonfluorescent blocks and not in the large fluorescent blocks.     

Differences between genetic and physical centromere distances in the case of two genes for male sterility in barley

Summary Linkage studies with thirty translocations (one of the two chromosomes involved being number 4) in relation to msg24 (chromosome 4) and thirteen translocations (one of the two chromosomes involved being number 6) in relation to msg6 (chromosome 6) show without exception close linkage for all combinations tested. The results indicate that both genes are located genetically in or close to the centromere regions of their chromosomes.Cytological analysis of two BTT stocks (balanced tertiary trisomics) ascertained the respective chromosome arms (both msg24 and msg6 on the short arms) and revealed marked differences between genetic and physical centromere distances. The reason is obviously the high content of centromeric heterochromatin occupying both the chromosome arms involved.     

Somatic hybrids and cybrids of Senecio fuchsii Gmel. (x) jacobaea L.

Summary In situ hybridisation and restriction fragment length polymorphism (RFLP) analysis were used to determine the relative location of the translocation breakpoint and the size of the integrated chromatin segment in hexaploid wheat-Lophopyrum translocation stocks. Three 7el₂-7D recombinant stocks were Robertsonian translocations, 7DS.7el. The remaining recombinant stock (KS10-2) was 7elS.7el-7DL and contained only the distal one-half of the long arm of 7D. The recombinant stock with 7el₁ (K11695) could be designated 7DS.7DL-7el where approximately the distal one-half of 7DL was replaced. RFLP analysis indicated that on the 7DL RFLP map the breakpoints for K11695 and KS10-2 are in different locations and that the two recombinants contain an overlapping region (a common region) of the Lophopyrum chromosome 7 in which Lr19, a leaf-rust resistant gene, is located. RFLP analysis also indicated that RFLP markers which mapped to within 1.5 cm of the centromere of chromosome 7D are located in the distal half of the long arm.     

Nonfluorescent Y chromosomes. Cytologic evidence of origin

Summary Twelve presumptive structurally altered Y chromosomes were studied with Q-, G-, G-11, C-, Cd, and lateral asymmetric banding techniques and were compared with normal X and Y chromosmes and with an abnormal [i(Yq)] Y chromosome that exhibited intact fluorescence. Significant to this work is the fact that the Y chromosome has a small block of Giemsa-11 heterochromatin adjacent to the centromere on the long arm, while the X chromosome does not, which allows a distinction between the X-and Y-derived chromosomes. Two of the twelve altered chromosomes of either X or Y origin are small nonfluorescent rings. Each ring has a G-11-positive band of heterochromatin at the centromere, confirming Y origin. Each of the normal-length nonfluorescent presumed Ys and a Y with a fluorescent band in the center have one G-11 band at the centromere and another at an equal distance from the end of the long arm, the bands also being Cd positive, indicating that these chromosomes are pseudodicentric. The likely mechanism of origin is a break at the distal bright heterochromatin/ euchromatin junction (or within the bright segment in the chromosome with the bright center band), fusion of the sister chromatids at the breakpoints, and loss of the distal segment.     

Localization of translocation breakpoints in somatic metaphase chromosomes of barley

Karyotype analyses based on staining by acetocarmine followed by Giemsa N-banding of somatic metaphase chromosomes of Hordeum vulgare L. were carried out on 61 reciprocal translocations induced by X-irradiation. By means of computer-based karyotype analyses all of the 122 breakpoints could be localized to defined sites or segments distributed over the seven barley chromosomes. The pre-definition of translocations with respect to their rearranged chromosome arms from other studies rendered it possible to define the break positions even in translocations having exchanged segments equal in size and the breakpoints located distally to any Giemsa band or other cytological marker. The breakpoints were found to be non-randomly spaced along the chromosomes and their arms. All breaks but one occurred in interband regions of the chromosomes, and none of the breaks was located directly within a centromere. However, short and long chromosome arms recombined at random. An improved tester set of translocations depicting the known break positions of most distal location is presented.     

Centromere-Independent Accumulation of Cohesin at Ectopic Heterochromatin Sites Induces Chromosome Stretching during Anaphase

Pericentric heterochromatin, while often considered as “junk” DNA, plays important functions in chromosome biology. It contributes to sister chromatid cohesion, a process mediated by the cohesin complex that ensures proper genome segregation during nuclear division. Long stretches of heterochromatin are almost exclusively placed at centromere-proximal regions but it remains unclear if there is functional (or mechanistic) importance in linking the sites of sister chromatid cohesion to the chromosomal regions that mediate spindle attachment (the centromere). Using engineered chromosomes in Drosophila melanogaster, we demonstrate that cohesin enrichment is dictated by the presence of heterochromatin rather than centromere proximity. This preferential accumulation is caused by an enrichment of the cohesin-loading factor (Nipped-B/NIPBL/Scc2) at dense heterochromatic regions. As a result, chromosome translocations containing ectopic pericentric heterochromatin embedded in euchromatin display additional cohesin-dependent constrictions. These ectopic cohesion sites, placed away from the centromere, disjoin abnormally during anaphase and chromosomes exhibit a significant increase in length during anaphase (termed chromatin stretching). These results provide evidence that long stretches of heterochromatin distant from the centromere, as often found in many cancers, are sufficient to induce abnormal accumulation of cohesin at these sites and thereby compromise the fidelity of chromosome segregation.     

Chromosomal Sites Necessary for Normal Levels of Meiotic Recombination in DROSOPHILA MELANOGASTER. I. Evidence for and Mapping of the Sites

Meiotic exchange was measured in females heterozygous for a normal sequence X chromosome and for each of eleven T(1;4)s and each of sixteen T(1;Y)s. The results indicate that the X chromosome can be divided into five intervals, such that heterozygosity for a breakpoint in one interval strongly suppresses exchange within that interval, but has little or no effect on exchange in other intervals. The boundaries between these intervals are identified and mapped to regions 3C4-6/7, 7A-7E, 11A and proximal to 18C on the standard salivary map; each boundary is located at (or within a small region containing) a major constriction (i.e., a block of intercalary heterochromatin).--Exchange was examined in females heterozygous for translocations broken within the constriction at 11A. The results imply that a boundary occupies only a subregion of the entire constriction and is subdivisible by translocation breakpoints. Several other properties of boundaries have been elucidated. Finally, the relationship of these data to a simple model of meiotic pairing proposed by I. Sandler (1956) and to the role of intercalary heterochromatin in the meiotic process is discussed.     

Physical mapping of nine Xq translocation breakpoints and identification of XPNPEP2 as a premature ovarian failure candidate gene

Women with balanced translocations between the long arm of the X chromosome (Xq) and an autosome frequently suffer premature ovarian failure (POF). Two "critical regions" for POF which extend from Xq13-->q22 and from Xq22-->q26 have been identified using cytogenetics. To gain insight into the mechanism(s) responsible for ovarian failure in women with X;autosome translocations, we have molecularly characterized the translocation breakpoints of nine X chromosomes. We mapped the breakpoints using somatic cell hybrids retaining the derivative autosome and densely spaced markers from the X-chromosome physical map. One of the POF-associated breakpoints in a critical region (Xq25) mapped to a sequenced PAC clone. The translocation disrupts XPNPEP2, which encodes an Xaa-Pro aminopeptidase that hydrolyzes N-terminal Xaa-Pro bonds. XPNPEP2 mRNA was detected in fibroblasts that carry the translocation, suggesting that this gene at least partially escapes X inactivation. Although the physiologic substrates for the enzyme are not known, XPNPEP2 is a candidate gene for POF. Our breakpoint mapping data will help to identify additional candidate POF genes and to delineate the Xq POF critical region(s).     

Telomere and centromere association tendencies in the human male metaphase complement

Summary Generalized distances between centromeres and between telomeres were statistically analyzed (x ² tests) in 100 trypsin-banded metaphase figures derived from normal males.Analysis of association tendencies on the first column of obtained c-c, p-p, q-p, and p-q histograms showed significant heterochromatin attraction not only between nonacrocentrics and acrocentrics but also between two nonacrocentric chromosome pairs (1 and 16). However since, not all c-heterochromatin-rich chromosomes were involved in associations (pair 5), and conversely, since chromosomes without an important centromeric heterochromatin block were involved in associations (pairs 8 and 11), it is probable that centromeric heterochromatin is not the only factor responsible for chromosome association. Moreover associations occur not only at the centromeres; in our circle analysis of the binding capacity of the telomeres or centromere of one chromosome pair with the telomeres or the centromeres of all other chromosome pairs, we also found preferential associations for T(4,13), T(9,15), T(11,15), T(13,19) T(15,19), T(17,18), T(17,22), and T(19,20).We therefore suggest that heterochromatin is not the only reason for chromosome association and that telomeres may also play an important part in this process.     

Preferential Breakpoints in the Recovery of Broken Dicentric Chromosomes in Drosophila melanogaster

We designed a system to determine whether dicentric chromosomes in Drosophila melanogaster break at random or at preferred sites. Sister chromatid exchange in a Ring-X chromosome produced dicentric chromosomes with two bridging arms connecting segregating centromeres as cells divide. This double bridge can break in mitosis. A genetic screen recovered chromosomes that were linearized by breakage in the male germline. Because the screen required viability of males with this X chromosome, the breakpoints in each arm of the double bridge must be closely matched to produce a nearly euploid chromosome. We expected that most linear chromosomes would be broken in heterochromatin because there are no vital genes in heterochromatin, and breakpoint distribution would be relatively unconstrained. Surprisingly, approximately half the breakpoints are found in euchromatin, and the breakpoints are clustered in just a few regions of the chromosome that closely match regions identified as intercalary heterochromatin. The results support the Laird hypothesis that intercalary heterochromatin can explain fragile sites in mitotic chromosomes, including fragile X. Opened rings also were recovered after male larvae were exposed to X-rays. This method was much less efficient and produced chromosomes with a strikingly different array of breakpoints, with almost all located in heterochromatin. A series of circularly permuted linear X chromosomes was generated that may be useful for investigating aspects of chromosome behavior, such as crossover distribution and interference in meiosis, or questions of nuclear organization and function.     

The X-autosome translocation in the common shrew (Sorex araneus L.): late replication in female somatic cells and pairing in male meiosis

Common shrews have an XX/XY₁Y₂ sex chromosome system, with the X chromosome being a translocation (tandem fusion) between the original X and an autosome; in males this autosome is represented by the Y₂ chromosome. From G-banded chromosomes, the Y₂ is homologous to the long arm and centromeric part of the short arm of the X. The region of the X that is homologous to the Y₂ and also the telomeric region of the short arm of the X were found to be early replicating in somatic cells from a female shrew after 5-bromo-2-deoxyuridine (BrdU) treatment in vitro. The remainder of the short arm of the X was shown to be late replicating. Electron microscopic examination of synaptonemal complexes in males at pachytene revealed pairing of the Y₂ axis with the long arm of the X, and Y₁ with the short arm. At early stages of pachytene, there is apparently extensive nonhomologous pairing between the X and Y₁. In essence, the short arm of the shrew X chromosome behaves like a typical eutherian X chromosome (it is inactivated in female somatic cells and is paried with the Y₁ during male meiosis) while the long arm behaves like an autosome (escapes the inactivation and pairs with the Y₂).